Thesis (Ph. D. in Molecular and Systems Toxicology and Pharmacology)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / Tetracyclines, a broad-spectrum class of antibiotics, were discovered in the late 1940s, and became widely used because of their important advantages: they are inexpensive, safe, demonstrate good oral absorption, and are active against a broad range of bacterial pathogens. Unfortunately, as with most antibiotics, the emergence of microbial resistance to tetracyclines has become a serious problem. Today, most genera examined have tetracycline-resistant isolates, although the percentage varies according to species and geographic location. Due to the emergence of resistance, tetracyclines are no longer the antibiotics of choice in treatment of many conditions, although they are still extensively used to treat a variety of bacterial infections. Substantial research efforts have been directed towards reversing tetracycline resistance in bacteria. This work describes the development of a novel anti-bacterial treatment for diseases caused by bacteria resistant to tetracycline. It was found that tetracycline-resistant bacteria expressing the TnlO gene of tetracycline resistance, upon induction with tetracycline, became extremely susceptible to destruction by the DNA-damaging anti-cancer drug cisplatin. Tetracycline-resistant bacteria grown in tetracycline and subsequently treated with cisplatin in the presence of tetracycline were killed about 10⁵-fold more effectively than wild-type bacteria and tetracycline-resistant bacteria not exposed to tetracycline. This phenomenon was observed in different strains of tetracycline-resistant E. coli. Other antibiotics tested with respective antibiotic-resistant bacteria did not produce the same effect of sensitization to cisplatin, suggesting a unique relationship among cisplatin, tetracycline and the tetracycline resistance / (cont.) gene. It was determined that levels of platinum DNA damage were higher in sensitized tetracycline-resistant cells than in wild-type cells, although total cellular platinum levels in sensitized tetracycline-resistant cells were not increased. At this time, the mechanism of increased DNA damage formation and the mechanism underlying sensitization to cisplatin are still matters of speculation. The experiments reported here, however, demonstrate that cells expressing the genes of tetracycline resistance actually became primary targets for destruction by cisplatin. Based on this study, it is suggested that the therapeutic power of the tetracyclines could be restored and enhanced by using a complementary drug that, in combination with tetracycline, would induce selective destruction of tetracycline-resistant bacteria. / by Doriana Froim. / Ph.D.in Molecular and Systems Toxicology and Pharmacology

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (leaves 61-67). / The distribution and generation of force within the myocardium during normal contractility is dictated by the tissue's underlying 3D myoarchitecture. The presence of disordered myoarchitecture may in turn constitute the pathological basis of impaired cardiac mechanics in numerous clinical conditions, such as the remodeling heart following myocardial infarction and cardiomyopathies. To investigate the multi-scale nature of architectural disarray in the setting of myocardial disease, a dual imaging approach consisting of diffusion spectrum magnetic resonance imaging (DSI) and high-speed multislice two-photon microscopy (TPM) was used. DSI is a technique that derives fiber orientation from directionality of proton diffusion, whereas TPM derives cellular alignment from an autocorrelation of 3D resolved images of cells and subcellular structure. Mesoscale tract representations of myofiber orientation are generated from similarly aligned diffusion or autocorrelation vectors. These methods were applied to study induced myocardial infarction in the rat and hypertrophic cardiomyopathy associated with deletion of the gene for myosin binding protein C (cMyBP-C) in the mouse. Normal cardiac muscle fiber alignment within the ventricular wall was characterized by a series of helical tracts transitioning from a lefthanded orientation in the subepicardium to circumferential in the mid-myocardium to righthanded in the subendocardium. Infarcted hearts displayed a fiber void in the infarct zone and an extension of both subepicardial and subendocardial fibers beyond the border zone. It's hypothesized that the growth of fibers contributes to the remodeling process and provides tensile strength to the myocardium during contraction. / (cont.) The hearts obtained from the cMyBP-C knockouts displayed significant myoarchitectural disarray characterized by a loss of voxel to voxel orientational coherence for fibers located from the mid-myocardium to subendocardium, resulting in a change in the transmural progression of remaining helical fibers. These observations suggest an association between cMyBP-C expression and cardiac fiber alignment, where variations in torsional rotation may constitute a mechanism for pump failure in hypertrophic cardiomyopathy. These results substantiate the use of multi-scale imaging methods to enhance understanding of molecular and cellular contributions to tissue mechanical function. / by Teresa T. Wang. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references. / Saccharomyces cerevisiae stimulates dendritic cells and represents a promising candidate for cancer immunotherapy development. Effective cross-presentation of antigen delivered to dendritic cells is necessary for successful induction of cellular immunity. Using a yeast vaccine model, we investigated the phagosome-to-cytosol pathway of cross-presentation. We demonstrate that the rate of antigen release from phagocytosed yeast directly affects cross-presentation efficiency, with an apparent time limit of about 25 min post-phagocytosis for antigen release to be productive. Antigen expressed on the yeast surface is cross-presented much more efficiently than antigen trapped in the yeast cytosol by the cell wall. The cross-presentation efficiency of yeast surface-displayed antigen can be increased by the insertion of linkers susceptible to cleavage in the early phagosome. Antigens indirectly attached to yeast through antibody fragments are less efficiently cross-presented when the antibody dissociation rate is extremely slow. Next, we present a yeast-based cancer vaccine approach that is independent of yeast's ability to express the chosen antigen, which is instead produced separately and conjugated to the yeast cell wall. The conjugation method is site-specific (based on the SNAP-tag) and designed to facilitate antigen release in the dendritic cell phagosome and subsequent translocation for cross-presentation. / (cont.) Phagosomal antigen release was further expedited through the insertion of the invariant chain ectodomain as a linker, which is rapidly cleaved by Cathepsin S. The dose of delivered antigen was increased in several ways: by using yeast strains with higher surface amine densities, by using yeast cell wall fragments instead of whole cells, and by conjugating multiple layers of antigen. The novel multi-layer conjugation scheme is site-specific and takes advantage of Sfp phosphopantetheinyl transferase, enabling the antigen dose to grow linearly. We show that whole yeast cells coated with one layer of the cancer-testis antigen NY-ESO-1 and yeast hulls bearing three layers were able to cross-prime naive CD8+ T cells in vitro, with the latter resulting in higher frequencies of antigen-specific cells after ten days. This cross-presentation-efficient antigen conjugation scheme is not limited to yeast and can readily be applied towards the development of other particulate vaccines. / by Shanshan W. Howland. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Includes bibliographical references (p. 211-223). / Glycosaminoglycans (GAGs) are a family of complex carbohydrates whose known biological roles have dramatically increased over the recent years. It is now becoming increasingly evident that sequence specific GAG-protein interactions play critical roles in cell growth, development, angiogenesis, cancer, anticoagulation and microbial pathogenesis. Therefore it is important to understand the specificity of glycan-protein interactions and how these specific interactions influence their structure-function relationships. This thesis addresses many challenges in this emerging area of glycomics, by taking an integrated approach that couples biophysical and biochemical methods with a bioinformatics framework to represent and process sequence information content in GAGs. With this motivation the thesis is divided into 4 components 1. Using heparin/heparan sulfate GAGs (HSGAGs) - fibroblast growth factor (FGF) as a model GAG-protein system, the first part focuses on determining the structural basis of FGF oligomerization, sequence specific FGF-HSGAG interactions and FGF-receptor (FGFR) interactions which collectively influence the specificity of HSGAG mediated FGF signaling. 2. The second part focuses on developing enzymatic tools for analysis of GAGs using chondroitinase B, HSGAG 2-0 sulfatase and 3-0 sulfotransferase as model enzymes. For each of these enzymes a theoretical model for the enzyme-substrate structural complex is developed and it is coupled with the site directed mutagenesis and biochemical studies to determine its catalytic mechanism and substrate specificity. 3. To deal with the heterogeneity and high information density of GAG sequences, an informatics based approach to decode GAG sequence information has been developed. / (cont.) A new property encoded nomenclature (PEN) computational framework has been formulated to encode and process information content in GAG sequences. The numerical nature of the PEN code facilitated the incorporation of diverse data sets from different analytical methods including mass spectrometry, electrophoresis and NMR as constraints to accurately determine the sequence of GAGs. Two practical methodologies for sequencing GAGs have been developed based on this approach. 4. The last part outlines the development of a powerful relational database that is capable of bridging sequence, structure and function information in glycomics. Thus this database and its associated computational tools to search and mine the data is an important resource for advancing glycomics. / by Rahul Raman. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / Cancer is a leading cause of death in the world. Much work has been done to study the role of inflammation in carcinogenesis. One hypothesis suggests that inflammation causes oxidative stress that induces damage to cellular targets, including DNA. The multistep model of cancer proposes that cancer is a genetic disease in which mutations are required in carcinogenesis. When this theory was championed, research focused on somatic mutations. The focus has broadened to include epigenetic mechanisms in changing gene expression. The association between chronic infection, chronic inflammation and increased cancer risk has been supported by epidemiologic studies. Data link chronic inflammation associated with infectious disease to increased cancer risk. Some examples of such infectious agents include hepatitis B virus, Schistosoma haemotobium, and Helicobacter pylori. One objective of this thesis was to investigate the role of inflammation in self-limiting infection. Additional objectives focus on evaluating a novel model of intestinal and extraintestinal cancer, and using immune regulating cells as treatment for intestinal cancer. For the first objective, a murine mutational analysis model was used to study infection with Citrobacter rodentium, an enteric bacterium that causes self-limiting hyperplasia and inflammation. Increased mutant frequency was observed in association with elevated levels of iNOS 13 days post infection. The second aim, to characterize a novel model of neoplasia, led to the discovery of basosquamous cancer in mice with intestinal tumors. Finally, Apc-[Min] mice, a model of intestinal neoplasia, were treated with T regulatory cells to investigate the role of these cells on tumor development. These / (cont.) cells were previously observed to have an anti-inflammatory and therapeutic effect on an infection-driven model of colon cancer. It was shown that T regulatory cells led to a decrease in the number of adenomas. In conclusion, it has been shown that self-limiting infection can increase mutant frequency. In addition, a novel model of intestinal and basosquamous cancer has been characterized, and a promising therapy for intestinal cancer has been validated. / by Jane Joo-hee Sohn. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2006. / Includes bibliographical references (p. 40-44). / The MilliF bioreactor offers great potential for the formation of i vivo-like liver tissue outside the body, making it a valuable tool for applications such as drug toxicity models and biosensors. Cell adhesion is an important factor in the maintenance of differentiated hepatocyte functions. Hepatocyte adhesion environments were examined in two settings: spheroid culture prior to seeding in the bioreactor and 2D surface culture methods that could be applied to the bioreactor scaffold. Spheroids were formed either by culturing in spinning suspension or on a static, non-adherent surface. In spheroid culture, the addition of extracellular matrix (ECM) signaling through the use of soluble Matrigel or adhesion protein-coated microspheres did not improve hepatocyte viability or function as assessed by liver-specific gene expression. These results suggest the importance of cell-cell rather than cell-surface interactions in maintaining hepatocytes. Optimal culturing of spheroids in spinning suspension without the ECM addition was found to be 3 days without media changes. 2D surfaces were treated with an adhesion peptide-conjugated comb polymer, preventing nonspecific cell adhesion and allowing attachment through the [alpha]₅[beta]₁ integrin. / (cont.) Varying the proportion of adhesion peptide presented to cells was found to regulate hepatocyte morphology and function; a surface with decreased hepatocyte spreading and liver-specific gene expression closer to in vivo was characterized. Immunoblotting for activated focal adhesion kinase (FAK) revealed that FAK signaling was not induced by attachment to the comb polymer surfaces. Immunostaining for other liver cell types demonstrated that the surface allowed hepatic stellate cell and Kupffer cell adhesion. / by Michael B. Wongchaowart. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / The extracellular matrix of tumors is a major barrier to the delivery of molecular medicine. We used fluorescence recovery after photobleaching combined with intravital microscopy to quantitate the transport properties of the tumor interstitium. We found that the presence of fibrillar collagen correlated with hindered diffusion in vivo, and also in vitro, in collagen gels prepared to mimic tumor extracellular matrix. Modification of the tumor collagen matrix directly with purified bacterial collagenase, or indirectly with relaxin treatment, resulted in increased diffusion coefficients of macromolecules within tumors in vivo. In order to quantitate the changes in collagen content and structure induced by relaxin treatment, we adapted and further developed the imaging technique of intravital second harmonic generation microscopy. Using second harmonic generation imaging in combination with a fluorescently labeled gene therapeutic vector, we demonstrated that the spread of these viral vectors within tumors is limited by the fibrillar collagen in the extracellular matrix. Matrix modification via the introduction of bacterial collagenase along with the initial virus injection resulted in a significant improvement in the range of viral distribution within the tumor. / (cont.) This resulted in an extended range of infection of cells within the tumor, and improved virus propagation, ultimately leading to enhanced therapeutic outcome. Thus, we show that fibrillar collagen is an important barrier to the distribution of molecular medicine within tumors, and that matrix modifying treatments can significantly enhance both vector distribution, as well as ultimately therapeutic response. / by Trevor David McKee. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 124-140). / Understanding the hepatotoxicity of drugs and chemicals is essential for progress in academic research, medical science and in the development of new pharmaceuticals. Studying hepatotoxicity in vitro is a challenging task because hepatocytes, the metabolically active cells of the liver, are very difficult to maintain in culture. After just 24 hours, the cells detach from the plate and die, and even if they survive they usually do not express the metabolic functions which they have in vivo. It has been observed by others that culturing hepatocytes between two layers of collagen type I maintains in vivo-like morphology and also many drug metabolizing enzymes for weeks. In spite of the research examining drug metabolism in collagen sandwiches, there are very few studies evaluating this system for investigating hepatotoxicity. We cultured primary rat hepatocytes in the collagen sandwich configuration and our goal was to optimize this system for long-term studies and to examine toxicity of a variety of hepatotoxins. By measuring secretions of urea and albumin, and P4501A activity, we determined the optimal cell density to be 50,000 cells/cm2. We also evaluated the need for epidermal growth factor (EGF) in our cultures, by comparing urea and albumin secretions in cultures grown with and without EGF. The cultures without EGF had significantly less secretion of both urea and albumin just two days after plating. Therefore, we decided to include EGF in the medium. The toxins we examined were aflatoxin B1, acetaminophen, carbon tetrachloride, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methane sulfonate (MMS), cadmium, vinyl acetate and dimethylformamide (DMF). The cells were sensitive to aflatoxin B1, MMS, MNNG and cadmium. However, they were / (cont.) immune to acetaminophen, carbon tetrachloride, vinyl acetate and DMF. Our Western Blots showed that CYP1A, 2B and 3A were maintained in the culture for a week, but CYP2E1 was lost gradually over time. CYP2E1 is also the primary metabolic enzyme for acetaminophen, carbon tetrachloride and DMF. Thus, it is possible that the lack of toxicity is due to the loss of the enzyme responsible for the metabolism of these compounds. Immunity to vinyl acetate suggests that carboxylesterase is also lost in culture, since this enzyme is the one which converts vinyl acetate to acetaldehyde. The metabolism of acetaminophen was also examined with liquid chromatography and mass spectrometry. Liquid chromatography showed that acetaminophen is metabolized primarily to the sulfate and glucuronide metabolites. In order to investigate whether the glutathione adduct was formed, we synthesized the adduct and determined its retention time with liquid chromatography and its fragmentation pattern with mass spectrometry. We isolated the fraction with the same retention time from the medium of acetaminophen-treated cells, and showed that it contains a peak with the same mass to charge ratio and fragmentation pattern as the glutathione adduct. We also examined the conditioned medium from the hepatocytes to investigate the secreted protein profile, which could potentially be used to find toxicity biomarkers. We were able to remove most of the albumin from the medium using an immuno-affinity column containing anti-albumin antibodies bound to protein A-agarose beads ... / by Dóra Farkas. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (p. 70-73). / Comb polymer, which consists of a hydrophobic poly(methyl methacrylate) (PMMA) backbone with hydrophilic hydroxy-poly(ethylene oxide) (HPOEM) side chains, is a tool that has many possible applications for the study of liver cell adhesion and signaling. This polymer has the unique properties of being cell resistant and chemically versatile such that various cell ligands can be coupled to its side chains. These properties allow adhesion through specific cell receptors to be studied without the effect of background adhesion to adsorbed proteins. By taking advantage of the ability to target specific receptors the comb polymer could be used as a powerful sorting tool. Sorting could be accomplished by finding cell type specific adhesion ligands. Several possible such ligands were screened. A ligand containing the tripeptide sequence RGD was found to elicit a strong cell adhesion response. However, this ligand is adherent to many cell types of the liver and would not be suitable for sorting purposes. Other cell type specific ligands tested showed little to no affinity for liver cell adhesion. Additionally, the comb was utilized to study α₅β₁ integrin-specific hepatocyte adhesion and the effect of Epidermal Growth Factor on adhesion. α₅β₁ integrin adhesion was mediated using a novel branched peptide, SynKRGD. This peptide consists of a linear peptide sequence containing RGDSP and the synergy site sequence PHSRN connected by the sequence GGKGGG. By utilizing the amine side group of Lysine a GGC branch was added. The terminal cysteine was used to conjugate SynKRGD to comb polymer surfaces using N-(p-Maleimidophenyl) isocyanate (PMPI) chemistry. EGF has a great potential to benefit the field of tissue engineering due to its influence on cell / (cont.) proliferation, migration, and differentiation. EGF is also known to have a de-adhesive effect in some cell types. Hepatocytes were studied on comb surfaces of variable SynKRGD densities with and without the presence of EGF in the media. Distinct morphological differences were observed for hepatocytes on substrates of varying adhesivity with and without the presence of EGF. EGF was found to have a de-adhesive effect on α₅β₁ integrin adhesion in hepatocytes. This effect became more pronounced as substrate adhesiveness increased. / by David Yin. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references. / (cont.) phosphoproteomics technology, IMAC/LC/MS/MS, [approximately] 200 phosphosites were identified from HT-29 cells, some of which were detected only from insulin-treated cells. Our phosphoproteomics approach also enabled us to detect alteration of both known and unknown phosphorylation states of apoptosis-related proteins at two time points during early apoptosis induced by tumor necrosis factor-α / Apoptosis, a physiologically regulated cell death, plays critical roles in development and immune system by maintaining tissue homeostasis. The thesis project investigates regulations of apoptosis in a human colon adenocarcinoma cell line, HT-29, exposed to diverse cellular stimuli, focusing on a specific protein as well as global level of proteins. The first part of the thesis demonstrated S-nitrosation of procaspase-9. S-nitrosation is a novel protein modification to regulate protein-protein interaction or protein activity. This modification has been implied to inactivate caspases. We could visualize S-nitrosation of an initiator caspase, procaspase-9, by enriching low-abundant procaspase-9 with immunoprecipitation and stabilizing S-nitroso-cysteine with biotin labeling. Nitric oxide synthase inhibitors and tumor necrosis factor-α (TNF-α) reduced the S-nitrosation level of procaspase-9, suggesting that S-nitrosation may be regulated by a nitric oxide synthase and denitrosation is likely a mechanism of apoptosis. The second part of the thesis is to examine survival effects of insulin on cells undergoing TNF-α-induced apoptosis. Insulin decreased the TNF-α-induced cleavage of key apoptotic mediators, caspases, and their substrates as well as apoptosis, in part, depending on phosphatidylinositol-3 kinase (PI-3K)/Akt pathway. One of protective mechanisms by insulin is likely to decrease the TNF-α-induced dissociation of a potent inhibitor of caspases, X-chromosome linked inhibitor of apoptosis protein (XIAP), from procaspase-9 via PI-3K/Akt pathway. Lack of phosphoproteomics data in HT-29 cells led the third part of the thesis to focus on investigating global level regulation of phosphoproteins during apoptosis. With a / by Ji-Eun Kim. / Ph.D.